How species with similar repertoires of protein-coding genes differ so markedly at the phenotypic level is poorly understood. By comparing organ transcriptomes from vertebrate species spanning ~350 million years of evolution, we observed significant differences in alternative splicing complexity between vertebrate lineages, with the highest complexity in primates. Within 6 million years, the splicing profiles of physiologically equivalent organs diverged such that they are more strongly related to the identity of a species than they are to organ type. Most vertebrate species-specific splicing patterns are cis-directed. However, a subset of pronounced splicing changes are predicted to remodel protein interactions involving trans-acting regulators. These events likely further contributed to the diversification of splicing and other transcriptomic changes that underlie phenotypic differences among vertebrate species.

Alternative splicing (AS) is a widespread mechanism underlying the generation of proteomic and regulatory complexity. However, which of the myriad of human AS events play important roles in disease is largely unknown. To identify frequently occurring AS events in lung cancer, we used AS microarray profiling and reverse transcription-PCR (RT-PCR) assays to survey patient-matched normal and adenocarcinoma tumor tissues from the lungs of 29 individuals diagnosed with non-small cell lung cancer (NSCLC). Of 5,183 profiled alternative exons, four displayed tumor-associated changes in the majority of the patients. These events affected transcripts from the VEGFA, MACF1, APP, and NUMB genes. Similar AS changes were detected in NUMB and APP transcripts in primary breast and colon tumors. Tumor-associated increases in NUMB exon 9 inclusion correlated with reduced levels of NUMB protein expression and activation of the Notch signaling pathway, an event that has been linked to tumorigenesis. Moreover, short hairpin RNA (shRNA) knockdown of NUMB followed by isoform-specific rescue revealed that expression of the exon 9-skipped (nontumor) isoform represses Notch target gene activation whereas expression of the exon 9-included (tumor) isoform lacks this activity and is capable of promoting cell proliferation. The results thus reveal widespread AS changes in NSCLC that impact cell signaling in a manner that likely contributes to tumorigenesis.Alternative splicing (AS), the process by which splice sites are differentially utilized to produce different mRNA isoforms, is a major step in the generation of proteomic and functional diversity in metazoans. At least 95% of human multiexon genes generate alternatively spliced transcripts, and the majority of these vary in level between different cell and tissue types (35,53). Previous studies have provided evidence that AS and the RNA binding proteins and other factors which control this process are often deregulated in cancers and other human diseases (7,14,15,17,49,51).In order to better understand the mechanisms underlying tumorigenesis, a critical goal is to identify consistent molecular changes underlying the initiation and progression of cancers. Such molecular changes represent promising candidates for diagnostic and therapeutic applications (15,39,56). Since AS often regulates subsets of genes that are not coregulated at the transcriptional level (31, 36), profiling of this layer of gene regulation has tremendous potential to identify molecular markers of cancer that are missed by other methods (3). For example, in the case of prostate cancer, it has been shown that AS signatures derived from microarray-based profiling are more reliable for diagnostic purposes than are signatures derived from mRNA expression profiling (61). Other studies have recently employed high-throughput reverse transcription-PCR (RT-PCR)-based screening of splicing changes in breast and ovarian tumor samples and have also revealed tumorassociated splicing signatures of potential diagnostic and prognostic v...

B67 The disruption of alternative splicing (AS) by either mutations in splicing sequences or changes in expression of splicing factors has been linked to many human diseases including cancer but the molecular changes associated with lung tumors are not well understood. Using our established quantitative AS microarray platform we have profiled matched normal and adenocarcinoma tissues from the lungs of 10 patients in order to identify changes at both the AS and transcriptional levels. These profiling experiments show a small set of exons that display pronounced and highly consistent changes between the normal and tumor tissues. This set is distinct from those genes showing changes at the transcriptional level. The results reveal how AS and transcription may be re-programmed during malignant transitions in the lung. Using a custom microarray with sets of exon body and splicing junction probes for profiling ~5000 human cassette alternative exons, we have identified 4 AS events that display pronounced inclusion level differences between normal and adenocarcinoma tissue in at least 80% of patients surveyed. These changes were confirmed by RT-PCR using the original 10 plus an additional 19 patient samples. Interestingly, all 4 of the alternative exons are located in genes that are linked to signaling pathways known to be deregulated in certain cancers. Moreover, these 4 AS events preserve frame and are conserved in mouse tissues, suggesting important functional roles. From the same dataset, a separate set of genes with transcript level changes were detected, consistent with previous findings that non-overlapping sets of genes are regulated at the AS and transcriptional levels, when comparing different tissues or corresponding tissues from different species. In addition to probe sets for profiling AS and transcript levels of the corresponding genes, our microarray contains probes to determine the transcript levels for 465 known and putative splicing factors. While significant changes in the expression levels of defined splicing factors were not detected, we observe changes in expression levels of 3 genes that contain RS domains, a feature of proteins that is predictive of a role in splicing. One of these genes is associated with the Notch pathway and the others are known tumor suppressor genes. We have confirmed that the change in AS for 1 of the target genes results in altered splicing at the protein level in addition to the RNA level changes. Work is in progress to characterize the functional role of this AS event using isoform-specific knockdown and overexpression. The increased expression of the exon-included protein isoform was evident in cell lines derived from both lung and colon cancer. We are currently determining if all 4 AS events occur in tumor tissue from breast and colon cancer patients. Also, we have expanded our profiling of human cancers by using higher density AS microarrays, and by using mRNA samples from additional normal and tumor-derived breast, colon and lung sources. Thus far, we have identified a set of conserved AS events that are consistently associated with lung adenocarcinomas. These are located in genes that function in signaling pathways that play important roles in tumorigenesis. Characterization of these AS events will advance our understanding of the role of splicing in human cancers and may also provide new targets for diagnostic applications as potential biomarkers. Citation Information: Cancer Prev Res 2008;1(7 Suppl):B67.